As proteínas que contem domínios ricos em leucina (LRR) mediam interações macromoleculares que estão envolvidas em muitos processos biológicos como infecção bacteriana em células hospedeiras e respostas imunológicas de plantas. Estudos anteriores em nosso laboratório identificaram um gene que codifica uma proteína contendo 6 LRRs (LaLRR17) em L. (L.) amazonensis. O LaLRR17 é um gene com expressão estágio regulada sendo abundantemente expresso na fase amastigota. Seqüências homólogas ao gene LaLRR17 foram encontradas em todas as espécies de Leishmania analisadas. Esse trabalho tem como objetivo a caracterização da proteína homóloga em L. (L.) major (LmLRR17). Anticorpos obtidos contra seqüências conservadas das proteínas LaLRR17 e LmLRR17 permitiram o estudo da abundância protéica em diferentes estágios do parasita. Curiosamente, a proteína LmLRR17 foi encontrada em maior abundância em promastigotas procíclicos em vez de amastigotas. Linhagens hiperexpressoras da proteína LmLRR17 ou expressoras da proteína LaLRR17 em fusão com o epitopo viral myc foram obtidas. As proteínas quiméricas foram expressas seguindo o mesmo padrão observado na cepa selvagem. O fenótipo desses mutantes foi avaliado mediante infecções de macrófagos in vitro. A hiperexpressão da proteína LmLRR17 em L. (L.) major não alterou o fenótipo da infecção in vitro. Por outro lado, a expressão da proteína heteróloga, LaLRR17, em promastigotas de L. (L.) major levou a incremento na virulência com maior número de células infectadas e de parasitas por célula. Esses resultados indicam que a expressão da proteína LmLRR17 em L. (L.) major é fortemente regulada. Esse trabalho também mostra que a expressão da proteína LaLRR17 em L. (L.) major leva a um aumento na infectividade. / Proteins containing leucine rich repeats (LRR) are known to be involved in macromolecular interactions in many processes such as signal transduction, cell-adhesion, RNA processing, apoptosis, disease resistance and immune response. A previous study in our laboratory identified a L. (L.) amazonensis gene encoding a protein containing 6 LRRs (LaLRR17). LaLRR17 is a stage-regulated gene expressed with increased abundance in the amastigote stage. Highly conserved homologues of LaLRR17 were found in all Leishmania species analyzed. Therefore, the aim of this study was to characterize the homologous protein of L. major (LmLRR17). Antibodies raised against peptide sequences common to LaLRR17 and LmLRR17 allowed the study of the steady-state protein abundance. Interestingly, LmLRR17 protein was found to be up-regulated in procyclic promastigotes, instead of amastigotes. Mutants of L. (L.) major overexpressing a myc-tagged version of LmLRR17 or of LaLRR17 protein were obtained. In these parasites, the chimeric proteins were expressed following the same pattern of expression observed in the wild type parasites. The phenotype of these mutants was assessed in vitro through macrophage infections. Overexpression of LmLRR17 protein in L. (L.) major resulted in an unaltered phenotype. On the other hand, overexpression of LaLRR17 in L. (L.) major induced an increase in virulence with a higher number of infected cells and intracellular parasites. These results indicate that the expression of LmLRR17 protein in L. major is tightly regulated and the expression of the heterologous LaLRR17 protein increased infectivity in vitro.

The goal of cancer chemotherapy is to develop effective, safe, and well-tolerated medications. The over-expression of certain receptors on cancer cell membrane provides a basis for active targeting by not only specific interaction between drug delivery system and cells, but also facilitated cellular uptake via receptor-mediated endocytosis. In this study, LDV oligomers up to six LDV repeating units were synthesized via solid phase peptide synthesis method, and evaluated as drug carrier as well as targeting moiety to deliver doxorubicin (Dox) to human malignant melanoma cells (A375), which over-express integrin α 4 β 1 . Cells expressing different levels of integrin α 4 β 1 or modulated using integrin α 4 -specific siRNA knock-down technique were verified by western blot and PCR. Magnetic beads with tripeptides LDV, VDL, or LNV on the surface were used in the binding specificity studies. Results verified that LDV was the minimally required ligand sequence for the specific binding to integrin α 4 β 1 , of which the interaction depends on the amount of integrin and can be utilized for the design of targeted drug delivery. The studies on A375 cells uptake of FITC-labeled LDV oligomers examined the effects of EDTA, temperature, endocytosis inhibitor, and competitive ligand. Cellular uptake mechanism was revealed to be temperature-dependent, receptor-mediated endocytosis, involving the specific interaction between LDV and integrin α 4 β 1 . The internalization extent of LDV monomer was the highest and was also inhibited to the most by the addition of free LDV when compared to other LDV oligomers. Cytotoxicity profiles of Dox-conjugated LDV oligomers were obtained on wild-type A375, integrin α4 knock-down A375, and normal human epithelial keratinocytes (NHEK) using SRB assay. A significant decrease (3∼6 folds) in the cytotoxicity of oligo(LDV)-Dox on A375 cells were observed when the integrin α4 expression was knocked down by ∼50%. Cytotoxicity further decreased on NHEK, which has the lowest integrin α4 expression among three cell lines. In contrast to oligo(LDV)-Dox, free Dox was not able to differentiate between cancerous and normal cells. This result demonstrated the potential of oligo(LDV) as targeting ligand. However, increase of repeating LDV unit did not lead to any apparent trend in cytotoxicity capacity. To facilitate the intracellular Dox release, hydrazone bond (HYD) was introduced between LDV and Dox. In vitro Dox release profiles in pH 6.0, 7.4, and rat plasma proved the pH-sensitivity of LDV-HYD-Dox. Cytotoxicity studies showed an increased cytotoxic effect of LDV-HYD-Dox when compared with LDV-Dox on wild-type A375 (2.5 times), knock-down A375 (1.5 times); while no significant difference in cytotoxicity on NHEK was observed. In vivo animal study supported the in vitro findings on LDV-HYD-Dox, which showed a significant inhibition of tumor growth and longest mice life span when compared to free Dox, poly(L,D,V)-Dox, and LDV-Dox, with averagely only ¼ of the tumor size and almost twice the life span of that from the free Dox group. In conclusion, based on the concept of specific interaction between LDV and integrin α 4 β 1 , oligo(LDV)-Dox targeted drug delivery system was developed and proved to be effective in the delivery of Dox to melanoma cells.

Duchenne Muscular Dystrophy (DMD) is an X-linked recessive, progressive muscle-wasting disease characterized by mutations in the dystrophin gene. Duchenne muscular dystrophy is the most common and most severe form of inherited muscle diseases, with an incidence of 1 in 3,500 male births1,2. Mutations in the dystrophin gene result in non-functional dystrophin or the complete absence of the protein dystrophin, resulting in necrosis and fibrosis in the muscle, loss of ambulation, cardiomyopathies, inadequate or failure of respiratory function, and decreased lifespan. Although there has been little research for effective nutritional strategies, dietary intervention may be effective as an adjuvant treatment. In this study, wild type (WT) and mdx animals were provided either a control or elevated branched chain amino acid (BCAA) diet nocturnally for 25 weeks to determine if the elevated BCAAs would attenuate muscle torque loss. Twenty-five weeks of chronic, elevated BCAA supplementation had no impact on muscle function measures. Interestingly, mdx and WT animals had the same torque responses in the low stimulation frequencies (1 Hz – 30 Hz) compared to higher stimulation frequencies. Tetanus was reached at a much lower stimulation frequency in mdx animals compared to WT animals (100 Hz vs +150 Hz). The mdx mouse consistently had more cage activity in the light cycle X- and Y-planes. Interestingly, animals on the BCAA diet increased X-, Y-, and Z-plane activity in the dark cycles at four weeks while animals on the control diet more Z-plane activity at 25 weeks, although not significant. All three BCAAs were elevated in the plasma at 25 weeks, although only Leu was significantly elevated. The BCAAs had no effect on. The diaphragm and skeletal muscle masses were larger in mdx animals, and WT animals had a significantly larger epididymal fat pad. The active state of BCKDC determined by phosphorylation of the E1α enzyme was greater in WT animals in white skeletal muscle, but not red skeletal muscle. Protein synthesis effectors of the mTORC1 signaling pathway and autophagy markers were similar among groups. Wild type animals had increased mTORC1 effectors and animals on the BCAA diet had decreased autophagy markers, although not significant. Although BCAAs did not affect muscle function, fibrosis, or protein synthesis effectors, this study illustrates the functionality of mdx muscles over time. It would be interesting to see how the different muscle fiber types are affected by DMD, noting the differences between the diaphragm, heart, red muscle, and white muscle fibrosis markers. Although there was no increase in mTORC1 effectors with an elevated BCAA diet, it would be interesting to determine muscle protein synthesis, myofibrillar protein synthesis, and total protein turnover in the mdx mouse with an elevated BCAA diet, although the dietary intervention started when mice arrived at 4 weeks of age, earlier intervention may be beneficial early in the disease process. / Doctor of Philosophy / Duchenne Muscular Dystrophy (DMD) is an X-linked recessive, progressive muscle-wasting disease characterized by mutations in the dystrophin gene. Duchenne muscular dystrophy is the most common and most severe form of inherited muscle diseases, with an incidence of 1 in 3,500 male births1,2. Mutations in the dystrophin gene result in non-functional dystrophin or the complete absence of the protein dystrophin, resulting in necrosis and fibrosis in the muscle, loss of movement and walking ability, cardiomyopathies, inadequate or failure of respiratory function, and decreased lifespan. Although there has been little research for effective nutritional strategies, dietary intervention may be effective as an adjuvant treatment and palliative care. The branched chain amino acids (BCAAs) are known to directly stimulate muscle protein synthesis by direct activation of the mechanistic target of rapamycin complex 1 (mTORC1). This study aimed to illustrate the differences between diseased and healthy mice and determine if BCAAs can reduce muscle torque loss. Twenty-five weeks of chronic, elevated BCAA supplementation had no impact on muscle function measures. Interestingly, mdx and WT animals had the same torque responses in the low stimulation frequencies (1 Hz – 30 Hz) compared to higher stimulation frequencies. Tetanus was reached at a much lower stimulation frequency in mdx animals compared to WT animals (100 Hz vs +150 Hz). The mdx mouse consistently had more cage activity in the light cycle X- and Y-planes. Interestingly, animals on the BCAA diet increased X-, Y-, and Z-plane activity in the dark cycles at four weeks while animals on the control diet more Z-plane activity at 25 weeks, although not significant. All three BCAAs were elevated in the plasma at 25 weeks, although only Leu was significantly elevated. The BCAAs had no effect on. The diaphragm and skeletal muscle masses were larger in mdx animals, and WT animals had a significantly larger epididymal fat pad. The active state of BCKDC determined by phosphorylation of the E1α enzyme was greater in WT animals in white skeletal muscle, but not red skeletal muscle. Protein synthesis effectors of the mTORC1 signaling pathway and autophagy markers were similar among groups. Wild type animals had increased mTORC1 effectors and animals on the BCAA diet had decreased autophagy markers, although not significant. Although BCAAs did not affect muscle function, fibrosis, or protein synthesis effectors, this study illustrates the functionality of mdx muscles over time. It would be interesting to see how the different muscle fiber types are affected by DMD, noting the differences between the diaphragm, heart, red muscle, and white muscle fibrosis markers. Although there was no increase in mTORC1 effectors with an elevated BCAA diet, it would be interesting to determine muscle protein synthesis, myofibrillar protein synthesis, and total protein turnover in the mdx mouse with an elevated BCAA diet, although the dietary intervention started when mice arrived at 4 weeks of age, earlier intervention may be beneficial early in the disease process.

Heart failure with preserved ejection fraction (HFpEF) is a complex syndrome associated with a high morbidity and mortality rate. Leucine supplementation has been demonstrated to attenuate cardiac dysfunction in animal models of cachexia and heart failure with reduced ejection fraction (HFrEF). So far, no data exist on leucine supplementation on cardiac function in HFpEF. Thus, the current study aimed to investigate the effect of leucine supplementation on myocardial function and key signaling pathways in an established HFpEF rat model. Female ZSF1 rats were randomized into three groups: Control (untreated lean rats), HFpEF (untreated obese rats), and HFpEF_Leu (obese rats receiving standard chow enriched with 3% leucine). Leucine supplementation started at 20 weeks of age after an established HFpEF was confirmed in obese rats. In all animals, cardiac function was assessed by echocardiography at baseline and throughout the experiment. At the age of 32 weeks, hemodynamics were measured invasively, and myocardial tissue was collected for assessment of mitochondrial function and for histological and molecular analyses. Leucine had already improved diastolic function after 4 weeks of treatment. This was accompanied by improved hemodynamics and reduced stiffness, as well as by reduced left ventricular fibrosis and hypertrophy. Cardiac mitochondrial respiratory function was improved by leucine without alteration of the cardiac mitochondrial content. Lastly, leucine supplementation suppressed the expression and nuclear localization of HDAC4 and was associated with Protein kinase A activation. Our data show that leucine supplementation improves diastolic function and decreases remodeling processes in a rat model of HFpEF. Beneficial effects were associated with HDAC4/TGF-β1/Collagenase downregulation and indicate a potential use in the treatment of HFpEF.

Nous avons démontré l’utilité du groupement protecteur tert-butylsulfonyle (N-Bus) pour la chimie des acides aminés et des peptides. Celui-ci est préparé en deux étapes, impliquant la réaction d’une amine avec le chlorure de tert-butylsulfinyle, suivie par l’oxydation par du m-CPBA, pour obtenir les tert-butylsulfonamides correspondants avec d’excellents rendements. Le groupement N-Bus peut être clivé par traitement avec 0.1 N TfOH/DCM/anisole à 0oC en 10h pour régénérer le sel d’ammonium. Une variété d’acides aminés N-Bus protégés ainsi que d’autres aminoacides peuvent alors être utilisés pour préparer divers dipeptides et tripeptides. A l’exception du groupe N-Fmoc, les conditions de déprotection du groupe N-Bus clivent également les groupements N-Boc, N-Cbz et O-Bn. Une déprotection sélective et orthogonale des groupes N-Boc, N-Cbz, N-Fmoc et O-Bn est également possible en présence du groupe protecteur N-Bus. Le nouvel acide aminé non-naturel (3R, 2R) 3–méthyl-D-leucine (β-Me-Leu) et son régioisomère 2-méthyle ont été synthétisés par ouverture d’une N-Ts aziridine en présence d’un excès de LiMe2Cu. Chacun des régioisomères du mélange (1:1,2) a été converti en la méthylleucine correspondante, puis couplé à l’acide D-phényllactique puis au motif 2-carboxyperhydroindole 4-amidinobenzamide en présence de DEPBT. Des élaborations ultérieures ont conduit à des analogues peptidiques non-naturels d’aeruginosines telles que la chlorodysinosine A. Les deux analogues ont ensuite été évalués pour leur activité inhibitrice de la thrombine et la trypsine. La présumée aeruginosine 3-sulfate 205B et son anomère β ont été synthétisés avec succès à partir de 5 sous-unités : la 3-chloroleucine, l’acide D-phényllactique, le D-xylose, le 2-carboxy-6-hydroxyoctahydroindole et l’agmatine. La comparaison des données RMN 1H et 13C reportées avec celles obtenues avec l’aeruginosine synthétique 205B révèle une différence majeure pour la position du groupe présumé 3'-sulfate sur l’unité D-xylopyranosyle. Nous avons alors synthétisés les dérivés méthyl-α-D-xylopyranosides avec un groupement sulfate à chacune des positions hydroxyles, afin de démontrer sans ambiguïté la présence du sulfate en position C-4' par comparaison des données spectroscopiques RMN 1H et 13C. La structure de l’aeruginosine 205B a alors été révisée. Une des étapes-clés de cette synthèse consiste en la formation du glycoside avec le groupe hydroxyle en C-6 orienté en axial sur la sous-unité Choi. Le 2-thiopyridylcarbonate s’est avéré une méthode efficace pour l’activation anomérique. Le traitement par AgOTf et la tétraméthylurée en solution dans un mélange éther-DCM permet d’obtenir l’anomère α désiré, qui peut alors être aisément séparé de l’anomère β par chromatographie / We have demonstrated the usefulness of tert-butylsulfonyl (N-Bus) protecting group in amino acid and peptide chemistry. It is formed in a 2-step procedure involving reaction of an amine with tert-butylsulfinyl chloride, followed by oxidation with m-CPBA to obtain the corresponding tert-butyl- sulfonamides in excellent yields. The N-Bus group can be cleaved to regenerate the corresponding amino salt in 0.1 N TfOH/DCM/anisole at 0 oC for 10 h. A variety of N-Bus protected amino acids and other common amino acids can be used to form dipeptides and tripeptides. With the exception of the N-Fmoc group, the conditions required for the N-Bus group cleavage also cleaved the N-Boc, N-Cbz and O-Bn groups. Selective and orthogonal deprotection of N-Boc, N-Cbz, N-Fmoc and O-Bn groups could be achieved in the presence of the N-Bus protecting group. The new unnatural amino acids (3R, 2R) 3–methyl-D-leucine (β-Me-Leu) and its 2-methyl regioisomer were synthesized by ring opening of an N-Ts aziridine intermediate with excess LiMe2Cu. The 1:1.2 mixture of regioisomers were each converted to the corresponding methyl leucines, then coupled to D-phenyllactic acid, followed by coupling with 2-carboxyperhydroindole 4-amidino-benzamide core in the presence of DEPBT. Further elaboration led to linear peptidic unnatural analogues of known aeruginosins such as chlorodysinosin A. The two analogues were also evaluated in enzymatic assays for their inhibitory activity against thrombin and trypsin. The presumed 3-sulfated aeruginosin 205B and its β–anomer were successfully synthesized from 5 subunits: 3-chloroleucine, D-phenyllactic acid, D-xylose, 2-carboxy-6-hydroxyoctahydroindole, and agmatine. Comparison of 1H and 13C NMR reported data with that of synthetic aeruginosin 205B revealed a disturbing discrepancy with regard to the position of the presumed 3'-sulfate on the D-xylopyranosyl unit. We synthesized methyl α-D-xylopyranosides with sulfates at each of the hydroxyl groups and conclusively demonstrated the the presence of a C-4'-sulfate by comparison of the 1H and 13C NMR spectroscopic data. Thus, the structure of aeruginosin 205B should be revised. One of the key steps in the synthesis is glycoside formation of the axially oriented C-6 hydroxyl group in the Choi subunit. The 2-thiopyridyl carbonate was a suitable method for anomeric activation, followed by treatment with AgOTf and tetramethylurea in ether-DCM solution to give the desired α-anomer, which was easily separable from the β-anomer by column chromatography.

La forme canadienne-française du syndrome de Leigh (LSFC) est une maladie métabolique associée à une déficience en cytochrome oxydase (COX) et caractérisée par des crises d’acidose lactique, menant à une mort prématurée. Les mécanismes qui sous-tendent l’induction des crises restent inconnus et il n’existe aucune thérapie efficace pour les prévenir. Cette étude vise à caractériser l'effet de facteurs métaboliques périphériques potentiellement altérés chez les patients LSFC sur la mort de lignées cellulaires issues de ces patients et de témoins puis, à identifier des agents thérapeutiques pouvant la prévenir. Nous postulons que (i) ces facteurs métaboliques induiront une mort prématurée des cellules de patients et que (ii) les interventions susceptibles de la prévenir pallieront les conséquences de la déficience en COX, soit la diminution des taux d’adénosine triphosphate (ATP) et l’augmentation du stress oxydant, du nicotinamide adénine dinucléotide (NADH) et des lipides toxiques. Un criblage de 8 facteurs sanguins et 10 agents thérapeutiques a été réalisé. Les paramètres mesurés incluent la nécrose, l’apoptose, l’ATP et l’activité de la COX. Les fibroblastes LSFC sont plus susceptibles à la mort par nécrose (39±6%) induite par du palmitate plus lactate, un effet associé à des niveaux d’ATP diminués (53±8%). La mort cellulaire est réduite de moitié par l’ajout combiné d’agents ciblant le NADH, l’ATP et les lipides toxiques, alors que l’ajout d’antioxydants l’augmente. Ainsi, un excès de nutriments pourrait induire la mort prématurée des cellules LSFC et, pour atténuer cette mort, il serait important de combiner plusieurs interventions ciblant différents mécanismes. / Leigh syndrome French-Canadian variant (LSFC) is a metabolic disease associated with cytochrome c oxidase (COX) deficiency and characterized by episodes of lactic acidosis, referred to as “crisis”, leading to death at an early age. The mechanisms underlying a crisis and its cellular consequences remain elusive, and there is no effective therapy. The aim of this study was to characterize the effect of peripheral metabolic factors that are potentially altered in patients with LSFC on their cells death and to identify therapeutic agents able to prevent them using cell-lineage from LSFC patients and controls. The hypothesis are that (i) these metabolic factors can induce premature death in patient cells, and (ii) interventions that could rescue these cells may target potential consequences of COX deficiency, namely low adenosine triphosphate (ATP), high nicotinamide adenine dinucleotide (NADH) and toxic lipids, as well as oxidative stress. A screening of 8 blood factors and 10 therapeutic agents was conducted in fibroblasts. Parameter measured included cell death by necrosis and apoptosis, as well as ATP level and COX activity. LSFC fibroblasts were more susceptible to necrosis (39±6%) induced by high palmitate plus lactate and this was associated with a lower ATP (53±8%). Cell death decreased 2-fold with combined interventions, which presumably act on NADH, ATP, and the accumulation of toxic lipids, but increased with antioxidants. Collectively, our results emphasize the importance of nutrient overload as a factor eliciting premature cell death in LSFC cells and of combining interventions acting through various mechanisms for cell death rescue.

Les protéines GILZ (Glucocorticoid-Induced Leucine Zipper) et TSC-22 (Transforming growth factor-beta Stimulated Clone-22) appartiennent à la famille de protéines TSC-22D (TSC-22 Domain). GILZ a été décrit précédemment comme étant induit au cours de la déprivation en interleukine-2 (IL-2) des lymphocytes de la lignée cellulaire CTLL-2, permettant ainsi de retarder leur apoptose. Le but de notre travail était de déterminer les rôles respectifs de GILZ et TSC-22 au cours de l’apoptose des cellules CTLL-2.Nos résultats ont permis de montrer que TSC-22 augmentait l’apoptose induite par la déprivation en IL-2 des cellules CTLL-2. Nous avons mis en évidence une augmentation de l’activation des caspases ainsi qu’une régulation positive de l’expression de BIM. Nous avons en outre montré que l’expression de GILZ, protéine anti-apoptotique, induite lors de la déprivation en IL-2, était régulée négativement en présence de TSC-22. Enfin, nous avons montré que l’expression de l’ARNm de gilz était régulée négativement par TSC-22, mais que la stabilité de son ARNm n’était pas modifiée.Notre travail a donc permis de montrer que TSC-22 accélère l’entrée en apoptose des lymphocytes T en régulant négativement l’expression de la protéine anti-apoptotique GILZ. / GILZ (Glucocorticoid-Induced Leucine Zipper) and TSC-22 (Transforming growth factor-beta Stimulated Clone-22) belong to the TSC-22D (TSC-22 Domain) family of proteins. GILZ has been previously shown to be induced upon interleukin-2 (IL-2) deprivation in the T-cell line CTLL-2, allowing cells to delay apoptosis. The aim of our study was to elucidate the respective roles of GILZ and TSC-22 during IL-2 deprivation-induced T-lymphocytes apoptosis.Our results demonstrated that TSC-22 increased CTLL-2 cells apoptosis induced upon IL-2 deprivation. We highlighted in TSC-22 expressing cells both an increase in caspases activation and BIM expression up-regulation. We also demonstrated that GILZ expression, an anti-apoptotic protein, known to be induced after IL-2 withdrawal, was down-regulated in the presence of TSC-22. Moreover, we showed that gilz mRNA expression was also significantly repressed, but gilz mRNA half-life was not modified.Altogether, these results suggest that, in T-cells, TSC-22 could behave as a repressor of GILZ expression, accelerating IL-2 deprivation-induced apoptosis.

Au cours du développement des végétaux, de l’établissement de l’identité cellulaire des premiers organes au guidage du tube pollinique, la communication cellule à cellule est d’une importance capitale. En réponse, les voies de signalisation moléculaires sont élaborées pour la perception d’un signal extérieur et la transduction en une réponse génique via une cascade intracellulaire. Les récepteurs kinases font partie des protéines perceptrices des stimuli et constituent chez les plantes une catégorie de protéines avec une occurrence considérable, mais dont très peu d’informations détaillées sont disponibles à ce jour. Une famille de récepteurs kinases chez Arabidopsis thaliana, AtORK11 (Arabidopsis thaliana Ovule Receptor Kinase 11), a été identifiée par orthologie à un récepteur spécifique aux ovaires chez une solanacéee sauvage, Solanum chacoense. La fonction présumée de cette famille de récepteurs kinases de type leucine-rich repeat, suggérée par son patron d’expression, implique les événements relatifs au développement des gamétophytes et à la reproduction. Afin de caractériser la fonction des quatre gènes de la famille (AtORK11a, AtORK11b, AtORK11c et AtORK11d) une stratégie d’analyse de mutants d’insertion de l’ADN-T et d’évaluation du mode d’action par complémentation bimoléculaire par fluorescence (BiFC) a été entreprise. Aucune fonction précise n’a pu être attribuée aux doubles mutants d’insertion, par contre la surexpression d’une construction dominante négative indique un rôle dans le développement gamétophytique. Il a aussi été démontré que les quatre récepteurs peuvent interagir par homodimérisation aussi bien que par hétérodimérisation. Une hypothèse de redondance fonctionnelle est ainsi mise à jour parmi la famille des gènes AtORK11. / Cell to cell communication is paramount during plant developmental processes, from cellular identity in early organogenesis to pollen tube guidance. In response to this requirement, molecular cell signalling is used to perceive an external signal and transduce the response by an intracellular signalling cascade leading to specific gene activation. The sensing protein is typically a receptor kinase, which will transduce the stimulus by phosphorylation of a cytoplasmic interaction partner. Although plant receptor kinases represent the largest protein kinase family, only handfuls are well characterized. By sequence identity (orthology), a family of leucine-rich repeat receptor kinases from Arabidopsis thaliana was identified as AtORK11 (Arabidopsis thaliana Ovule Receptor Kinase 11). Based upon previous results from its ortholog in Solanum chacoense, the ovary- specific ScORK11 receptor kinase, we hypothesized that members of the AtORK11 receptors would be involved in gametophyte development and reproduction. In order to characterize the role of the four family members (AtORK11a, AtORK11b, AtORK11c and AtORK11d), a T-DNA insertional mutant strategy was undertaken, as well as bimolecular fluorescence complementation assays (BiFC). No precise function could be assigned to the double mutants although a dominant negative strategy revealed an involvement in gametophytic development. It was also shown that all of the receptors could form homodimers as well as heterodimers in a heterologous system, suggesting high functional redundancy for the AtORK11 family.

Nous avons démontré l’utilité du groupement protecteur tert-butylsulfonyle (N-Bus) pour la chimie des acides aminés et des peptides. Celui-ci est préparé en deux étapes, impliquant la réaction d’une amine avec le chlorure de tert-butylsulfinyle, suivie par l’oxydation par du m-CPBA, pour obtenir les tert-butylsulfonamides correspondants avec d’excellents rendements. Le groupement N-Bus peut être clivé par traitement avec 0.1 N TfOH/DCM/anisole à 0oC en 10h pour régénérer le sel d’ammonium. Une variété d’acides aminés N-Bus protégés ainsi que d’autres aminoacides peuvent alors être utilisés pour préparer divers dipeptides et tripeptides. A l’exception du groupe N-Fmoc, les conditions de déprotection du groupe N-Bus clivent également les groupements N-Boc, N-Cbz et O-Bn. Une déprotection sélective et orthogonale des groupes N-Boc, N-Cbz, N-Fmoc et O-Bn est également possible en présence du groupe protecteur N-Bus. Le nouvel acide aminé non-naturel (3R, 2R) 3–méthyl-D-leucine (β-Me-Leu) et son régioisomère 2-méthyle ont été synthétisés par ouverture d’une N-Ts aziridine en présence d’un excès de LiMe2Cu. Chacun des régioisomères du mélange (1:1,2) a été converti en la méthylleucine correspondante, puis couplé à l’acide D-phényllactique puis au motif 2-carboxyperhydroindole 4-amidinobenzamide en présence de DEPBT. Des élaborations ultérieures ont conduit à des analogues peptidiques non-naturels d’aeruginosines telles que la chlorodysinosine A. Les deux analogues ont ensuite été évalués pour leur activité inhibitrice de la thrombine et la trypsine. La présumée aeruginosine 3-sulfate 205B et son anomère β ont été synthétisés avec succès à partir de 5 sous-unités : la 3-chloroleucine, l’acide D-phényllactique, le D-xylose, le 2-carboxy-6-hydroxyoctahydroindole et l’agmatine. La comparaison des données RMN 1H et 13C reportées avec celles obtenues avec l’aeruginosine synthétique 205B révèle une différence majeure pour la position du groupe présumé 3'-sulfate sur l’unité D-xylopyranosyle. Nous avons alors synthétisés les dérivés méthyl-α-D-xylopyranosides avec un groupement sulfate à chacune des positions hydroxyles, afin de démontrer sans ambiguïté la présence du sulfate en position C-4' par comparaison des données spectroscopiques RMN 1H et 13C. La structure de l’aeruginosine 205B a alors été révisée. Une des étapes-clés de cette synthèse consiste en la formation du glycoside avec le groupe hydroxyle en C-6 orienté en axial sur la sous-unité Choi. Le 2-thiopyridylcarbonate s’est avéré une méthode efficace pour l’activation anomérique. Le traitement par AgOTf et la tétraméthylurée en solution dans un mélange éther-DCM permet d’obtenir l’anomère α désiré, qui peut alors être aisément séparé de l’anomère β par chromatographie / We have demonstrated the usefulness of tert-butylsulfonyl (N-Bus) protecting group in amino acid and peptide chemistry. It is formed in a 2-step procedure involving reaction of an amine with tert-butylsulfinyl chloride, followed by oxidation with m-CPBA to obtain the corresponding tert-butyl- sulfonamides in excellent yields. The N-Bus group can be cleaved to regenerate the corresponding amino salt in 0.1 N TfOH/DCM/anisole at 0 oC for 10 h. A variety of N-Bus protected amino acids and other common amino acids can be used to form dipeptides and tripeptides. With the exception of the N-Fmoc group, the conditions required for the N-Bus group cleavage also cleaved the N-Boc, N-Cbz and O-Bn groups. Selective and orthogonal deprotection of N-Boc, N-Cbz, N-Fmoc and O-Bn groups could be achieved in the presence of the N-Bus protecting group. The new unnatural amino acids (3R, 2R) 3–methyl-D-leucine (β-Me-Leu) and its 2-methyl regioisomer were synthesized by ring opening of an N-Ts aziridine intermediate with excess LiMe2Cu. The 1:1.2 mixture of regioisomers were each converted to the corresponding methyl leucines, then coupled to D-phenyllactic acid, followed by coupling with 2-carboxyperhydroindole 4-amidino-benzamide core in the presence of DEPBT. Further elaboration led to linear peptidic unnatural analogues of known aeruginosins such as chlorodysinosin A. The two analogues were also evaluated in enzymatic assays for their inhibitory activity against thrombin and trypsin. The presumed 3-sulfated aeruginosin 205B and its β–anomer were successfully synthesized from 5 subunits: 3-chloroleucine, D-phenyllactic acid, D-xylose, 2-carboxy-6-hydroxyoctahydroindole, and agmatine. Comparison of 1H and 13C NMR reported data with that of synthetic aeruginosin 205B revealed a disturbing discrepancy with regard to the position of the presumed 3'-sulfate on the D-xylopyranosyl unit. We synthesized methyl α-D-xylopyranosides with sulfates at each of the hydroxyl groups and conclusively demonstrated the the presence of a C-4'-sulfate by comparison of the 1H and 13C NMR spectroscopic data. Thus, the structure of aeruginosin 205B should be revised. One of the key steps in the synthesis is glycoside formation of the axially oriented C-6 hydroxyl group in the Choi subunit. The 2-thiopyridyl carbonate was a suitable method for anomeric activation, followed by treatment with AgOTf and tetramethylurea in ether-DCM solution to give the desired α-anomer, which was easily separable from the β-anomer by column chromatography.

Au cours du développement des végétaux, de l’établissement de l’identité cellulaire des premiers organes au guidage du tube pollinique, la communication cellule à cellule est d’une importance capitale. En réponse, les voies de signalisation moléculaires sont élaborées pour la perception d’un signal extérieur et la transduction en une réponse génique via une cascade intracellulaire. Les récepteurs kinases font partie des protéines perceptrices des stimuli et constituent chez les plantes une catégorie de protéines avec une occurrence considérable, mais dont très peu d’informations détaillées sont disponibles à ce jour. Une famille de récepteurs kinases chez Arabidopsis thaliana, AtORK11 (Arabidopsis thaliana Ovule Receptor Kinase 11), a été identifiée par orthologie à un récepteur spécifique aux ovaires chez une solanacéee sauvage, Solanum chacoense. La fonction présumée de cette famille de récepteurs kinases de type leucine-rich repeat, suggérée par son patron d’expression, implique les événements relatifs au développement des gamétophytes et à la reproduction. Afin de caractériser la fonction des quatre gènes de la famille (AtORK11a, AtORK11b, AtORK11c et AtORK11d) une stratégie d’analyse de mutants d’insertion de l’ADN-T et d’évaluation du mode d’action par complémentation bimoléculaire par fluorescence (BiFC) a été entreprise. Aucune fonction précise n’a pu être attribuée aux doubles mutants d’insertion, par contre la surexpression d’une construction dominante négative indique un rôle dans le développement gamétophytique. Il a aussi été démontré que les quatre récepteurs peuvent interagir par homodimérisation aussi bien que par hétérodimérisation. Une hypothèse de redondance fonctionnelle est ainsi mise à jour parmi la famille des gènes AtORK11. / Cell to cell communication is paramount during plant developmental processes, from cellular identity in early organogenesis to pollen tube guidance. In response to this requirement, molecular cell signalling is used to perceive an external signal and transduce the response by an intracellular signalling cascade leading to specific gene activation. The sensing protein is typically a receptor kinase, which will transduce the stimulus by phosphorylation of a cytoplasmic interaction partner. Although plant receptor kinases represent the largest protein kinase family, only handfuls are well characterized. By sequence identity (orthology), a family of leucine-rich repeat receptor kinases from Arabidopsis thaliana was identified as AtORK11 (Arabidopsis thaliana Ovule Receptor Kinase 11). Based upon previous results from its ortholog in Solanum chacoense, the ovary- specific ScORK11 receptor kinase, we hypothesized that members of the AtORK11 receptors would be involved in gametophyte development and reproduction. In order to characterize the role of the four family members (AtORK11a, AtORK11b, AtORK11c and AtORK11d), a T-DNA insertional mutant strategy was undertaken, as well as bimolecular fluorescence complementation assays (BiFC). No precise function could be assigned to the double mutants although a dominant negative strategy revealed an involvement in gametophytic development. It was also shown that all of the receptors could form homodimers as well as heterodimers in a heterologous system, suggesting high functional redundancy for the AtORK11 family.